Resea rc h A r tic le
Relationship between postprandial endotoxemia in nonobese postmenopausal women and diabetic nonobese postmenopausal women Gaffar Sarwar Zaman, Fawzia Zaman1
Department of Clinical Biochemistry, Government College of Medicine, King Khalid University, Abha, Kingdom of Saudi Arabia, 1Head of Operations and Quality Manager Ekopath Metropolis, Guwahati, Assam, India Address for correspondence: Dr. Gaffar Sarwar Zaman, Department of Clinical Biochemistry, Government College of Medicine, King Khalid University, Abha, Kingdom of Saudi Arabia. E-mail: [email protected]
Abstract Background: We hypothesised that nonobese postmenopausal women (NoPoW) and diabetic NoPoW (DNoPoW) may be independently associated with postprandial endotoxemia. Materials and Methods: NoPoW and DNoPoW were evaluated for weight, eating habits, physical activity, body circumferences, fasting plasma glucose level, postprandial plasma glucose level, and insulin level. The lipopolysaccharide (LPS) levels and circulating LPS-binding protein (LBP) were determined in serum at fasting, 1 h, 2 h, 3 h, and 4 h after meal intake and their levels were co-related in 80 NoPoW and 80 DNoPoW. Results: Both DNoPoW group and NoPoW group showed a significant increase (P < 0.05) in LPS levels and circulating LBP in plasma after the meal intake, interestingly the increase was higher in the DNoPoW group. Conclusions: Elevated LPS and circulating LBP were associated significantly with DNoPoW group and NoPoW, especially after a meal intake. These findings suggested a role of LPS and LBP in postprandial systemic inflammation in DNoPoW group. Prospective studies are needed to confirm these results. Key words: Endotoxemia, lipopolysaccharide, lipopolysaccharide-binding protein, postmenopause
INTRODUCTION Lipopolysaccharide (LPS) is an endotoxin (molecular weight >100,000 Daltons) and is composed of two major parts, the hydrophobic lipid A portion and the hydrophilic polysaccharide portion (commonly called the “O” region). Circulating endotoxin may derive from bacteria or gut microflora causing either overt acute infections or common chronic inflammatory conditions or cardiometabolic abnormalities including obesity, insulin resistance, and diabetes.[1] Endotoxin circulates in the plasma of healthy human subjects at low concentrations (between 1 and 200 pg/Ml).[2-5] However as the human gut Access this article online Quick Response Code:
Website: www.jnsbm.org
DOI: 10.4103/0976-9668.149098
89
is host to 100 trillion commensal organisms, which together contribute to an enteric reservoir of 1 g LPS, it is hypothesized that most of the circulating endotoxin may derive from the gut and that a small amount of commensally derived LPS may cotransit with dietary fat from the gut after a high-lipid meal, which thereby increases plasma endotoxin concentrations postprandially. The increased cardiovascular disease (CVD) risk after menopause seems to be associated with the emergence of the features of metabolic syndrome.[6,7] In fact, women with type 2 diabetes mellitus (T2DM), compared with age-matched nondiabetic women, exhibit several-fold higher rates of death-related to coronary artery disease, with event rates nearly identical to those observed in T2DM men.[8] Traditional cardiovascular risk factors cannot completely account for these sex differences in cardiovascular mortality.[9] Hence, more studies are needed to understand the precise influence of menopause in the risk for CVD, especially in diabetic patients, in order to achieve effective preventative and disease management
Journal of Natural Science, Biology and Medicine | January 2015 | Vol 6 | Issue 1
Zaman and Zaman: PP Endotoxemia in diabetic and nondiabetic postmenopause
strategies to reduce the CVD risk particularly in postmenopausal women. Estrogen exerts cardioprotective action by maintaining a high level of high-density lipoprotein cholesterol and lowering the low-density lipoprotein cholesterol and triglycerides.[10-13] Loss of this protection after menopause may, therefore, be responsible for increased risk of developing CVD in postmenopausal women.[14-17] Increases in body weight are associated with greater risks of type 2 diabetes,[18-20] and weight gain in postmenopausal women are of special concern.[21] Estrogen has remarkable effects on body fat distribution, and the decreased estrogen production after menopause is associated with increased total body fat,[22,23] especially in the central/abdominal region.[24,25]
MATERIALS AND METHODS Patients inclusion and exclusion criteria
This study was conducted in accordance with the ethical rules of the Helsinki Declaration. The study was approved by the Ethics Committee of the hospital, and all women gave written informed consent. Prior to the study, participants were informed that their confidentiality would be maintained, and consent was obtained. Eighty nonobese postmenopausal women (NoPoW) and 80 diabetic NoPoW (DNoPoW) were selected for the study. For the 80 NoPoW patients were excluded if they had CVD, arthritis, acute inflammatory disease, infectious disease, renal disease, were receiving treatment for hyperlipidemia or diabetes or were taking medications that could influence gastric emptying or the absorption time. For the 80 DNoPoW, all the above criteria except diabetes cases were excluded. Preparation of patients and sample collection
On the morning of the visit, blood pressure, weight, and height were measured, and compliance with dinner instructions was verified with a questionnaire. After that, each participant underwent a structured examination, which included an interview. Height, weight, waist circumference (WC) and hip measurements, a fasting venipuncture, and sequential determination of serum lipids were done. Height and weight were measured to the nearest 0.5 cm and 0.1 kg, respectively. Body mass index (BMI) was calculated as weight (kilogram) divided by height (in meter) squared. WC was determined to the nearest 0.1 cm using a measuring tape positioned at the midpoint between the lowest rib and the iliac crest and hips were measured at the largest gluteal circumference. These measurements were used to calculate the waist-to-hip ratio. Blood pressure was measured using a standard mercury sphygmomanometer. Blood samples were obtained from the antecubital vein and placed in vacutainer tubes. Postprandial blood samples were taken 1, 2, 3, and 4 h after the end of the study meal. Samples
were centrifuged; serum was collected and stored at 20°C until analyzed. The diagnosis of DM was based on WHO criteria,[26] that is, a fasting plasma glucose level >7.0 mmol/L or >126 mg/dL, or a 2-h postprandial plasma glucose level >11.1 mmol/L or >200 mg/dL on more than one occasion, with symptoms of diabetes. Serum LPS concentrations were measured by endotoxin assay, based on a Limulus amebocyte extract with a chromogenic Limulus amebocyte lysate (LAL) assay (QCL-1000, Lonza Group Ltd.). Samples were diluted in pyrogen-free water and heated at 70°C for 10 min to inactivate endotoxin-neutralizing agents that inhibit the activity of endotoxin in the LAL assay. Internal control of recovery calculation was included in the assessment. All samples were tested in duplicate. The endotoxin content was expressed as endotoxin units (EU)/ mL. Exhaustive care was taken to avoid environmental endotoxin contamination and all material used for sample preparation, and the test was pyrogen-free. Plasma LPSbinding protein (LBP) levels were determined by a sandwich enzyme-linked immunosorbent assay Technology. Plasma samples were diluted at least 200 times and assayed according to the manufacturer’s instructions. The assay has a sensitivity of 0.2 ng/ml. The intra-assay and inter assay coefficients of variation were
Relationship between postprandial endotoxemia in nonobese postmenopausal women and diabetic nonobese postmenopausal women Gaffar Sarwar Zaman, Fawzia Zaman1
Department of Clinical Biochemistry, Government College of Medicine, King Khalid University, Abha, Kingdom of Saudi Arabia, 1Head of Operations and Quality Manager Ekopath Metropolis, Guwahati, Assam, India Address for correspondence: Dr. Gaffar Sarwar Zaman, Department of Clinical Biochemistry, Government College of Medicine, King Khalid University, Abha, Kingdom of Saudi Arabia. E-mail: [email protected]
Abstract Background: We hypothesised that nonobese postmenopausal women (NoPoW) and diabetic NoPoW (DNoPoW) may be independently associated with postprandial endotoxemia. Materials and Methods: NoPoW and DNoPoW were evaluated for weight, eating habits, physical activity, body circumferences, fasting plasma glucose level, postprandial plasma glucose level, and insulin level. The lipopolysaccharide (LPS) levels and circulating LPS-binding protein (LBP) were determined in serum at fasting, 1 h, 2 h, 3 h, and 4 h after meal intake and their levels were co-related in 80 NoPoW and 80 DNoPoW. Results: Both DNoPoW group and NoPoW group showed a significant increase (P < 0.05) in LPS levels and circulating LBP in plasma after the meal intake, interestingly the increase was higher in the DNoPoW group. Conclusions: Elevated LPS and circulating LBP were associated significantly with DNoPoW group and NoPoW, especially after a meal intake. These findings suggested a role of LPS and LBP in postprandial systemic inflammation in DNoPoW group. Prospective studies are needed to confirm these results. Key words: Endotoxemia, lipopolysaccharide, lipopolysaccharide-binding protein, postmenopause
INTRODUCTION Lipopolysaccharide (LPS) is an endotoxin (molecular weight >100,000 Daltons) and is composed of two major parts, the hydrophobic lipid A portion and the hydrophilic polysaccharide portion (commonly called the “O” region). Circulating endotoxin may derive from bacteria or gut microflora causing either overt acute infections or common chronic inflammatory conditions or cardiometabolic abnormalities including obesity, insulin resistance, and diabetes.[1] Endotoxin circulates in the plasma of healthy human subjects at low concentrations (between 1 and 200 pg/Ml).[2-5] However as the human gut Access this article online Quick Response Code:
Website: www.jnsbm.org
DOI: 10.4103/0976-9668.149098
89
is host to 100 trillion commensal organisms, which together contribute to an enteric reservoir of 1 g LPS, it is hypothesized that most of the circulating endotoxin may derive from the gut and that a small amount of commensally derived LPS may cotransit with dietary fat from the gut after a high-lipid meal, which thereby increases plasma endotoxin concentrations postprandially. The increased cardiovascular disease (CVD) risk after menopause seems to be associated with the emergence of the features of metabolic syndrome.[6,7] In fact, women with type 2 diabetes mellitus (T2DM), compared with age-matched nondiabetic women, exhibit several-fold higher rates of death-related to coronary artery disease, with event rates nearly identical to those observed in T2DM men.[8] Traditional cardiovascular risk factors cannot completely account for these sex differences in cardiovascular mortality.[9] Hence, more studies are needed to understand the precise influence of menopause in the risk for CVD, especially in diabetic patients, in order to achieve effective preventative and disease management
Journal of Natural Science, Biology and Medicine | January 2015 | Vol 6 | Issue 1
Zaman and Zaman: PP Endotoxemia in diabetic and nondiabetic postmenopause
strategies to reduce the CVD risk particularly in postmenopausal women. Estrogen exerts cardioprotective action by maintaining a high level of high-density lipoprotein cholesterol and lowering the low-density lipoprotein cholesterol and triglycerides.[10-13] Loss of this protection after menopause may, therefore, be responsible for increased risk of developing CVD in postmenopausal women.[14-17] Increases in body weight are associated with greater risks of type 2 diabetes,[18-20] and weight gain in postmenopausal women are of special concern.[21] Estrogen has remarkable effects on body fat distribution, and the decreased estrogen production after menopause is associated with increased total body fat,[22,23] especially in the central/abdominal region.[24,25]
MATERIALS AND METHODS Patients inclusion and exclusion criteria
This study was conducted in accordance with the ethical rules of the Helsinki Declaration. The study was approved by the Ethics Committee of the hospital, and all women gave written informed consent. Prior to the study, participants were informed that their confidentiality would be maintained, and consent was obtained. Eighty nonobese postmenopausal women (NoPoW) and 80 diabetic NoPoW (DNoPoW) were selected for the study. For the 80 NoPoW patients were excluded if they had CVD, arthritis, acute inflammatory disease, infectious disease, renal disease, were receiving treatment for hyperlipidemia or diabetes or were taking medications that could influence gastric emptying or the absorption time. For the 80 DNoPoW, all the above criteria except diabetes cases were excluded. Preparation of patients and sample collection
On the morning of the visit, blood pressure, weight, and height were measured, and compliance with dinner instructions was verified with a questionnaire. After that, each participant underwent a structured examination, which included an interview. Height, weight, waist circumference (WC) and hip measurements, a fasting venipuncture, and sequential determination of serum lipids were done. Height and weight were measured to the nearest 0.5 cm and 0.1 kg, respectively. Body mass index (BMI) was calculated as weight (kilogram) divided by height (in meter) squared. WC was determined to the nearest 0.1 cm using a measuring tape positioned at the midpoint between the lowest rib and the iliac crest and hips were measured at the largest gluteal circumference. These measurements were used to calculate the waist-to-hip ratio. Blood pressure was measured using a standard mercury sphygmomanometer. Blood samples were obtained from the antecubital vein and placed in vacutainer tubes. Postprandial blood samples were taken 1, 2, 3, and 4 h after the end of the study meal. Samples
were centrifuged; serum was collected and stored at 20°C until analyzed. The diagnosis of DM was based on WHO criteria,[26] that is, a fasting plasma glucose level >7.0 mmol/L or >126 mg/dL, or a 2-h postprandial plasma glucose level >11.1 mmol/L or >200 mg/dL on more than one occasion, with symptoms of diabetes. Serum LPS concentrations were measured by endotoxin assay, based on a Limulus amebocyte extract with a chromogenic Limulus amebocyte lysate (LAL) assay (QCL-1000, Lonza Group Ltd.). Samples were diluted in pyrogen-free water and heated at 70°C for 10 min to inactivate endotoxin-neutralizing agents that inhibit the activity of endotoxin in the LAL assay. Internal control of recovery calculation was included in the assessment. All samples were tested in duplicate. The endotoxin content was expressed as endotoxin units (EU)/ mL. Exhaustive care was taken to avoid environmental endotoxin contamination and all material used for sample preparation, and the test was pyrogen-free. Plasma LPSbinding protein (LBP) levels were determined by a sandwich enzyme-linked immunosorbent assay Technology. Plasma samples were diluted at least 200 times and assayed according to the manufacturer’s instructions. The assay has a sensitivity of 0.2 ng/ml. The intra-assay and inter assay coefficients of variation were
